Azole derivative and pharmaceutical composition

ABSTRACT

A novel azole derivative of the general formula (I):    &lt;IMAGE&gt;  (I)  wherein R1 and R2 are, independently from each other, a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, x1 and x2 are, independently from each other, a hydrogen or halogen atom, or hydroxyl, cyano, or trifluoromethyl group, and Y is CH or a nitrogen atom, a process for the production thereof, and a pharmaceutical composition containing the same as the active ingredient.

This is a Divisional of application Ser. No. 08/137,045 filed on Oct.14, 1993, which was derived from International Patent Application No.PCT/JP93/00181 filed on Feb. 12, 1993.

TECHNICAL FIELD

The present invention relates to a novel azole derivative, a process forthe production thereof, and a pharmaceutical composition containing theazole derivative as an active ingredient (more particularly, anantifungal agent or an aromarase inhibitor).

BACKGROUND ART

The recent development of pharmaceuticals and the advancement of medicaltechniques conquered many diseases. On the other hand, such treatmentcaused the depression in the immune systems. The depression became amajor cause of the increase in patients susceptible to infection. Thesepatients suffered at a high rate from deep-seated fungal diseases ofopportunistic infections such as candidiasis, aspergillosis, andcryptococcosis. The measure to solve the problem became serious.Therefore, active research has been under way to develop drugs moresuperior than the conventional antifungal agents. For example, JapaneseUnexamined Patent Publication (Kokai) No. 3-187464 discloses an azolederivative having a cyclohexanol ring and azole ring as basicstructures. Such antifungal agents exhibit an antifungal activity byfunctioning on the cytochrome p450 in the fungus and inhibiting theproduction of ergosterol which is a constituent element of cell walls.Further, it is known that because the above antifungal agents functionon the cytochrome p450, some of them exhibit an activity to inhibitaromarase [J. Med. Chem., 33 (11), 2933-2942 (1990)].

From the results of the research on azole derivatives having.applicability to broader fields and exhibiting a more superiorantimicrobial activity, the inventors of the present invention found anovel azole derivative which has a low toxicity, and which exhibitsactivity against many fungi, and activity to inhibit an aromarase. Thepresent invention is based on the above findings.

DISCLOSURE OF THE INVENTION

Therefore, the present invention relates to a compound of the generalformula (I): ##STR2## wherein R₁ and R₂ are, independently from eachother, a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, X₁and X₂ are, independently from each other, a hydrogen or halogen atom,or hydroxyl, cyano or trifluoromethyl group, and Y is CH or a nitrogenatom, or a salt thereof. In the above general formula (I), preferably,R₁ and R₂ are, independently from each other, a hydrogen atom or analkyl group having 1 to 2 carbon atoms, X₁ and X₂ are, independentlyfrom each other, a hydrogen or halogen atom, or hydroxyl group, and Y isCH or a nitrogen atom.

Further, the present invention relates to a compound of the generalformula (III): ##STR3## wherein R₁, R₂, X₁, and X₂ have the samemeanings as above, or a salt thereof.

Further, the present invention relates to a compound of the generalformula (IV): ##STR4## wherein R₁, R₂, X₁, and X₂ have the same meaningsas above, or a salt thereof.

Still further, the present invention relates to a process for theproduction of a compound of the general formula (I) characterized byreducing a compound of the general formula (V): ##STR5## wherein R₁, R₂,X₁, and X₂ have the same meanings as above, to obtain the compound ofthe general formula (IV), reacting the resulting compound of the generalformula (IV) and an S-ylide compound to obtain the compound of thegeneral formula (III), and reacting the resulting compound of thegeneral formula (III) and a compound of the general formula (II):##STR6## wherein M is a metal ion and Y has the same meaning as above.

Still further, the present invention relates to a pharmaceuticalcomposition, particularly, an antifungal agent or an aromataseinhibitor, characterized by containing a compound of the general formula(I) or a salt thereof.

BEST MODE FOR CARRYING OUT THE INVENTION

In the general formulae (I), (III), (IV), or (V), the alkyl group of 1to 4 carbon atoms as the group R₁ or R₂ is a methyl, ethyl,straight-chain or branched propyl, or straight-chain or branched butylgroup. The halogen atom is a chlorine, bromine, iodine, or fluorineatom. Further, in the general formula (II), the metal ion M is an alkalimetal, for example, sodium or potassium.

The azole derivative of the general formula (I) [hereinafter sometimesreferred to as the present compound (I) or the present azole compound(I)] may be manufactured, for example, from a compound of the generalformula (V) by a process comprising the following steps (a), (b), and(c).

(a) The compound of the general formula (V) is reduced to obtain thecompound of the general formula (IV) [hereinafter sometimes referred toas the present cyclohexanone compound (IV)].

(b) The resulting present cyclohexanone compound (IV) is reacted with anS-ylide compound to obtain the compound of the general formula (III)[hereinafter sometimes referred to as the present oxolane compound(III)].

(c) The present oxolane compound (III) is reacted with the1,2,4-triazole or imidazole of the general formula (II), therebyobtaining the present azole compound (I).

As the diluents which may be used in the series of reactions in thesteps (a), (b), and (c), there may be mentioned hydrocarbons, such asbenzene, toluene, xylene or hexane; halogenated hydrocarbons, such asmethylene chloride, chloroform or carbon tetrachloride; alcohols, suchas methanol, ethanol or isopropyl alcohol; ethers, such as diethylether, diisopropyl ether, tetrahydrofuran or dioxane; and further, ethylacetate, acetonitrile, acetone, dimethylformamide, or dimethylsulfoxide.

Further, the series of reactions in steps (a), (b), and (c) may also becarried out in the presence of a base or acid, in addition to the abovediluent. As the bases which can be used, there may be mentionedcarbonates of alkali metals, such as sodium or potassium carbonate;hydroxides of alkali metals, such as sodium or potassium hydroxide;alcoholates of alkali metals, such as sodium methylate, sodium ethylateor potassium t-butylate; hydride of alkali metals, such as sodium orpotassium hydride; alkylates of alkali metals, such as n-butyl lithium;and further, triethylamine, or pyridine. Further, as the acids, theremay be mentioned inorganic acids, such as hydrochloric, hydrobromic,hydriodic, or sulfuric acid; organic acids, such as formic, acetic,butyric, or p-toluenesulfonic acid.

The reducing reaction in the step (a) may be carried out in the presenceof, for example, a platinum catalyst, palladium carbon and potassiumcarbonate or palladium carbon, and a conventional oxidizing agent, suchas Jone's reagent. That is, the compound of the general formula (V) isdissolved in an organic solvent (for example, alcohol), a palladiumcarbon catalyst is added, and the reducing reaction is carried out forabout 5 to 24 hours in a hydrogen stream. Then, the solvent is removed,an organic solvent is further added, Jone's reagent or the like isadded, and the oxidation reaction is carried out. After the reaction isceased by adding alcohol (for example, isopropyl alcohol), the resultingproduct is poured into ice water, extracted with a solvent (for example,ethyl acetate, diethyl ether). The product is dried over sodium sulfateor the like to remove the solvent, and the residue is purified by columnchromatography to obtain the desired present cyclohexanone compound(IV). In the cyclohexene ring of the compound of the general formula (Vused as the starting material and the cyclohexane ring of the presentcyclohexanone compound (IV), the configurations at the ring carbon atomto which the groups R₁ and R₂ are bonded and the chiral ring carbon atomto which the phenyl group is bonded are not limited. Each of the opticalisomers having any configuration or a mixture thereof may be used. Theoptical isomers may be resolved and purified by chromatography (forexample, thin layer chromatography, column high performance liquidChromatography, or optical isomer separation column chromatography) anda general method of optical isomer separation. The compound of thegeneral formula (V) may be prepared by the method described in, forexample, Monalsh Chem. 9, 1043, 1960.

The present cyclohexanone compound (IV) includes the optical isomers ofthe following general formulae (IVA), (IVA'), (IVB), and (IVB'):##STR7## wherein R₁, R₂, X₁, and X₂ have the same meanings as above.

The purification is performed by recrystallization, silica gel columnchromatography, or the like. In the step (b), the configurations at theasymmetric carbon atoms in the rings of the starting compound and thefinal compound are not limited. Each of the pure optical isomers havingany configuration or mixtures thereof may be used. The optical isomersmay be resolved and purified in the same manner as above. The presentoxolane compound (III) includes optical isomers of the following generalformulae [III(t-(+))], [III(t-(-))], [III(c-(-)], and [III(c-(+))]:##STR8## wherein R₁, R₂, X₁, and X₂ have the same meanings as above.

The reaction of the step (c) may be performed by dissolving the1,2,4-triazole or imidazole of the general formula (II) in theabove-mentioned diluent, and adding the present oxolane compound (III),in the presence of the above-mentioned base as occasion demands, oralternatively, by dissolving the present oxolane compound (III) in thediluent, and then adding the 1,2,4-triazole or imidazole of the generalformula (II). The reaction temperature is about 0° to 150° C.,preferably about 40° to 120° C., and the reaction time is about 0.5 to24 hours, preferably about 1 to 10 hours. After the above-mentionedreaction is completed, the reaction mixture is cooled and extracted inice water with an organic solvent, such as ethyl acetate, chloroform,methylene chloride, diethyl ether, or benzene to separate the organiclayer. Then, the organic layer is washed with water and dried. Further,the solvent is removed under reduced pressure and the resulting residueis purified to obtain the desired present azole compound (I). Thepurification is performed by recrystallization, silica gel columnchromatography, or the like. In the step (c), the configurations at thechiral carbon atoms of the starting compound and the final compound arenot limited. Each of the pure optical isomers having any configurationor mixtures thereof may be used. The present azole compound (I) includesthe optical isomers of the following general formulae [I(t-(+))],[I(t-(-))], [I(c-(-)], and [I(c-(+))]: ##STR9## wherein R₁, R₂, X₁, andX₂ have the same meanings as above.

The present azole compound (i) exhibits a pharmacological activity, inparticular an antifungal activity, and is useful for overcoming fungalinfections in mammals, including humans. Therefore, the presentinvention relates also to a pharmaceutical composition, particularly,antifungal agent, containing the present azole compound (I) or apharmaceutically or veterinarily acceptable salt thereof, and apharmaceutically or veterinarily acceptable diluent or carrier. Theantifungal agent of the present invention is useful for the treatment oflocal fungal infections in humans, particularly those caused by fungibelonging to the genera Candida, Trichophyton, Microsporum, orEpidermophyton, or infections of the mucous membranes caused by Candidaalbicans (for example, oral Candidiasis or vaginal Candidiasis).Further, the antifungal agent of the present invention may also be usedfor the treatment of systemic fungal infections caused by Candidaalbicans, Cryptococcus neoformans, Aspergillus fumigatus, or fungibelonging to the genera Coccidioides, Paracoccidioides, Histoplasma, orBlastomyces.

The antifungal activity of the present azole compound (I) in a livingbody was evaluated by determining the minimum inhibitory concentration(MIC) in vitro for the fungi belonging to the genera Candida,Cryptococcus, Aspergillus, and Trichophyton.

The present azole compound also exhibits an aromatase inhibitoryactivity, and an antitumor activity along with the aromarase inhibitoryactivity. Therefore, the present invention also relates to an aromataseinhibitory agent, particularly an antitumor agent, containing thepresent azole compound (I) or a pharmaceutically or veterinarilyacceptable salt thereof, and a pharmaceutically or veterinarilyacceptable diluent or carrier. Aromarase is an enzyme which aromatizesthe ring A of many steroid hormones in the course of metabolism.Further, many cancers (for example, breast cancer, cancer of the uterus,prostate cancer, pancreatic cancer, ovarian cancer, or the like) dependon the steroid hormone having the aromatic ring A. Therefor, the presentazole compound (I) exhibits an antitumor activity on these cancers.

The aromarase inhibitory activity was determined by the method of Covey,D. F. et al., [BBRC (1), 81-86, 1988]. That is, the aromarase inhibitoryactivity was determined as the 50% inhibitory concentration IC₅₀ valueof the enzyme activity of the compound to be tested, whereupon the IC₅₀value of the present azole compound (I) was not more than 10⁻⁶ M.

The present azole compound (I) may be mixed with a carrier generallyacceptable for pharmaceutical compositions, and used in the form ofvarious formulations. These compositions may be formulated into units ofdosage containing about 1 to 600 mg, more preferably about 5 to 500 mg,of the present azole compound (I) in the form of dosage. The presentazole compound (I) may be a salt, such as a sulfate, nitrate, orcitrate. The pharmaceutical composition of the present invention may beadministered orally, endermically, or intravenously.

When treating adults, it is suitable to administer about 0.1 to 100mg/kg in one dosage or divide into several dosages. However, the actualdosage is determined by the physician with reference to the age of theindividual patient, the seriousness of the symptoms, and the route ofadministration, so the range of the dosage mentioned above may besometimes exceeded, but these cases are also included in the scope ofthe present invention. The acute toxicity (LD₅₀) of the present azolecompound (I) was found using ICR mice to be over 500 mg/kg, so it isapparent that the present azole compound (I) is safe.

EXAMPLES

The present invention now will be further illustrated by, but is by nomeans limited to, the following examples. In the following examples, theNMR was measured using a JNM-GSX500 (Nihon Denshi), the infraredabsorption spectra were measured using a Nihon Bunko A-202 apparatus,and the optical rotation was measured using a Nihon Bunko automaticpolarimeter DIP-360.

Example 1 [1] Preparation of(2,2-dimethyl)-5-(4-fluorophenyl)-5-cyclohexene-1-one [V-1].

1-bromo-4-fluorobenzene (7.06 g, 40 mmole) was dissolved in 20 ml oftetrahydrofuran, then the resulting solution was stirred at -78° C. inan argon gas stream. N-butyl lithium (25.2 ml, 1.6 mole solution) wasadded slowly and the solution was allowed to stand for 15 minutes. Asolution of 5 g (40 mmole) of 4,4-dimethyl-2-cyclohexene-1-one in 20 mlof tetrahydrofuran was slowly added to the above solution, and then, theresulting solution was allowed to stand for 30 minutes. Thereafter, asaturated aqueous solution of ammonium chloride was added, the wholesolution was poured into ice water, and extracted with ethyl acetate.The organic layer was dried over sodium sulfate to remove the solvent.20 ml of acetone was added to the residue, until the Jone's reagentpreviously prepared no longer changed color (until it no longer becamegreen). After 30 minutes, the reaction was ceased by adding isopropylalcohol. The whole was poured into ice water, and the crude product wasextracted with ethyl acetate. The organic layer was dried over sodiumsulfate to the solvent was removed by evaporation. The residue waspurified by column chromatography (10% ethyl acetate/hexane) to obtainthe above-titled compound [V-1] (7.8 g).

¹ H-NMR (δ ppm; CDCl₃) 1.17 (s, 6H), 1.97 (t, 2H, J=11.9 Hz), 2.75 (t,2H, J=11.9 Hz), 6.30 (s, 1H), 7.10, 7.55 (each m, 2H)

[2] Preparation (2,2-dimethyl)-5-(4-fluorophenyl)-cyclohexane-1-one[IV-1]

The compound [V-1] (4 g, 18.3 mole) obtained in the Example 1[1] wasdissolved in 20 ml of ethyl alcohol. After 100 mg of 10% palladiumcarbon was added, the solution was stirred overnight in a hydrogen gasstream. The-next morning, the reaction solution was filtered, and thesolvent was evaporated. Then, 10 ml of acetone was added to the residue,and Jone's reagent previously prepared was added until the reagent nolonger changed in color (until no longer becoming green). After 30minutes, the reaction was ceased by adding isopropyl alcohol. Thesolution was poured into ice water and extracted with ethyl acetate. Theorganic layer was dried over sodium sulfate, and the solvent wasevaporated. The residue was purified by column chromatography (10% ethylacetate/hexane) to obtain the above-titled compound [IV-1] (3.27 g).

¹ H-NMR (δ ppm; CDCl₃)

1.12, 1.24 (each s, 3H) , 1.65 to 2.05 (m, 4H) , 2.47, 2.71 (each m,1H), 2.96 (m, 1H), 7.02, 7.18 (each m, 2H) IR (νmax; KBr): 1690, 1602

Example 2 [1] Preparation oft-(+)-(2,2-dimethyl)-5-(4-fluorophenyl)-cyclohexane-1,1'-oxolane[III(t-(+))-1] andt-(-)-(2,2-dimethyl)-5-(4-fluorophenyl)-cyclohexane-1,1'-oxolane[III(t-(-))-2]

To a mixture of 5 ml of dimethyl sulfoxide and 10 ml of tetrahydrofuran,499.2 mg (20.8 mmole) of sodium hydride which had been washed withn-hexane was suspended. 4.425 g (20.8 mmole) of trimethylsulfoniumiodide was added to the suspension under cooling with ice. The mixturewas stirred for 30 minutes, then 5 ml of a dimethyl sulfoxide solutionof 4.2729 g (19.4 mmole) of the compound [v-1] obtained in the Example1[1] was added dropwise slowly, whereupon the reaction solution turnedlight yellow. The solution was stirred overnight at room temperature.Then, the reaction was ceased by pouring the solution into 100 ml of icewater. The crude product was extracted with diethyl ether (100 ml×2 and50 ml×1). The collected ether layers were washed with a saturated salinesolution and dried over anhydrous sodium sulfate, then concentratedunder reduced pressure to obtain an oily crude product (4.312 g). Thecrude product was purified by silica gel chromatography, whereupon theabove-titled compounds [III(t(-(+))-1] and [III(t-(-))-2] (0.8726 g)were obtained as a racemic mixture mainly in the second peak withn-hexane:ethyl acetate (80:1).

¹ H-NMR (δ ppm; CDCl₃) 0.80 (s, 3H), 1.15 (s, 3H), 1.23 (m, 1H), 1.5-1.8(m, 4H), 2.29 (t, 1H), 2.49 (d, 1H, J=4.59 Hz), 2.92 (d, 1H, J=4.59 Hz),2.72 (m, 1H), 6.98 (m, 2H), 7.16 (m, 2H)

[2] Preparation of (2,2-dimethyl)-5S-(4-fluorophenyl)-1R-(1H-1,2,4-triazole-1-ylmethyl)-cyclohexane-1-ol[I(t-(+))-1]

The racemic mixture (872.6 mg, 3.72 mmole) obtained in the Example 2[1]was dissolved in 10 ml of dimethyl formamide. A sodium salt of1,2,4-triazole was added to the solution. The whole was heated andstirred in an argon atmosphere for 24 hours at 80° C. After the reactionwas completed, the reaction solution was poured into ice water (50 ml)and the obtained crude product was extracted with diethyl ether (100ml×2 and 50 ml×1). The collected ether layers were washed with 20 ml ofa saturated saline solution and dried over anhydrous sodium sulfate,then concentrated under reduced pressure, whereupon a crude product(1.113 g) was obtained. The crude product was purified by silica gelchromatography (n-hexane:ethyl acetate=1:1) to obtain the desiredproduct as a racemic mixture (0.7249 g). The racemic mixture wasresolved by chiracell OG, whereupon the above-titled compound[I(t-(+))-1] (30.2 mg) was obtained in the second peak upon separationat 3 ml/min using 5% isopropyl alcohol/n-hexane solution as a movingphase at 40° C.

Melting point=155° to 156° C. Optical rotation (c=0.1; methanol)=+40°;Chemical purity=92%

Example 3 [1] Preparation of(2,2-dimethyl)-5R-(4-fluorophenyl)-1S-(1H-1,2,4-triazole-1-ylmethyl)cyclohexane-1-ol[I-(t-(-))-2]

The racemic mixture obtained in the Example 2[2] was separated under theconditions shown in the Example 3[2] using chiracell OG, whereupon theabove-titled compound [I(t-(-))-2] (26.1 mg) was obtained in the firstpeak.

Melting point=156° to 157° C. Optical rotation (c=0.1; methanol)=-40°;Chemical purity=91.1%

Example 4 [1] Preparation of c-(-)-(2,2-dimethyl)5-(4-fluorophenyl)-cyclohexane-1,1'-oxolane [III(c-(-))-3] andc-(+)-(2,2-dimethyl)-5-(4-fluorophenyl)-cyclohexane-1,1-oxolane[III(c-(+))-4]

Trimethylsulfoxoniumiodide (5.831 g, 26.5 mmole) was placed in a 100 mleggplant type flask and dissolved in 30 ml of dimethyl sulfoxide. 629.1mg (26.2 mmole) of sodium hydride which had been washed with n-hexanewas added to the above solution. The solution was stirred at roomtemperature for 1 hour, whereupon the reaction solution becametransparent. A solution of the above compound [IV-1] (3.85 g, 17.5mmole) dissolved in 10 ml of dimethyl sulfoxide was added to thereaction solution. The whole was stirred at 10° C. for 5 minutes and atroom temperature for 5 hours, whereupon the starting materialdisappeared. The resulting solution was dried over anhydrous sodiumsulfate, and concentrated under reduced pressure by a rotary evaporatorto obtain the crude product. The crude product was purified by silicagel chromatography, whereupon the above-titled compounds [III(C-(-)))-3]and [III(C-(+))-4] were obtained as a racemic mixture in the firstfraction of the n-hexane ethyl acetate (20:1).

¹ H-NMR (δ ppm; CDCl₃) 0.79 (s, 3H), 1.16 (s, 3H), 1.23 (d, d, 1H),1.51-1.58 (m,4H), 2.28 (t, 1H), 2.42 (d, 1H, J=4.59 Hz), 2.84 (d, 1H,J=4.59 Hz), 2.90 (m, 1H), 6.97 (m, 2H), 7.1 (m, 2H)

[2] Preparation of(2,2-dimethyl)-5S-(4-fluorophenyl)-1S-(1H-1,2,4-triazole-1-ylmethyl)cyclohexane-1-ol[I(C-(-))-3]

The racemic mixture (4.00 g, 17.5 mmole) obtained from the Example 4[1]was dissolved in 35 ml of dimethylformamide. 12.65 g (26.5 mmole) ofsodium salt of 1,2,4-triazole was added to the solution, and the wholewas heated at 90° C. under stirring in an argon atmosphere for 4 hours.After the reaction was completed, the reaction solution was poured into35 ml of ice water and the crude product was extracted with diethylether (150 ml×1, 50 ml×1, and 30 ml×1). The collected ether layers werewashed with distilled water (20 ml×1) and dried over anhydrous sodiumsulfate, then were concentrated under reduced pressure to obtain thedesired racemic crude product. The racemic mixture was dissolved inethyl acetate and the mother solution was purified by silica gelchromatography with ethyl acetate, whereupon the purified product of theracemic mixture was obtained. The racemic mixture was separated usingchiracell OD (separation conditions, moving phase; isopropyl ether:n-hexane=15:85, 1.2 ml/min) to obtain the above-titled compound[I(c-(-))-3] in the second peak.

Melting point=139° to 140° C. Optical rotation (c=0.01; methylalcohol)=-41.7° Chemical purity=99.6%, Optical purity=99.4%

Example 5 [1] Preparation of(2,2-dimethyl)-5R-(4-fluorophenyl)-1R-(1H-1,2,4-triazole-1-ylmethyl)cyclohexane-1-ol[I-(C-(+)-4]

A racemic mixture was obtained by the method described in the Example4[2]. The resulting racemic mixture was separated using theabove-mentioned chiracell OD used in the Example 4[2] under the sameconditions to obtain the desired above-titled compound [I(c(+))-4] inthe first peak.

Melting point=138° to 139° C. Optical rotation (c=0.01; methylalcohol)=+43.0° Chemical purity=98.7%; Optical purity=100%

Example 6 [1] Preparation of(2,2-dimethyl)-5-(4-chlorophenyl)-5-cyclohexene-1-one [V-2]

1-bromo-4-chlorobenzene (7.66 g, 40 mmole) and 25 ml of driedtetrahydrofuran were placed in a 100 ml eggplant type flask. The wholewas stirred in an argon atmosphere at room temperature and cooled to-78° C. Then, 25 ml (40 mmole) of n-butyl lithium was slowly added andthe whole was stirred for 30 minutes. Then, 4.96 g (40 mmole) of4,4-dimethyl-2-cyclohexene-1-one dissolved in dried tetrahydrofuran wasadded dropwise. After 90 minutes, 10 ml of a saturated aqueous solutionof ammonium chloride was added, and the whole was poured into 10 ml ofice water. Thereafter, extraction was performed with diethyl ether (100ml×2 and 50 ml×1). The ether layers were washed with a saturated salinesolution and dried over anhydrous sodium sulfate, then was dried underreduced pressure. The crude product was purified by silica gelchromatography (n-hexane: ethyl acetate=4:1). The resulting yellow oilyproduct (19.48 g; 82.29 mmole) was added to a 200 ml eggplant typeflask, and 85 ml of acetone was added. After stirring, 25 ml of Jone'sreagent was added dropwise. After 15 minutes, the reaction was ceased byadding 85 ml of cold water. Thereafter, the crude product was extractedwith diethyl ether (100 ml×2), the ether layers were washed with asaturated saline solution, and dried over anhydrous sodium sulfate, anddried under reduced pressure. The residue was purified by silica gelchromatography (n-hexane:ethyl acetate=8:1) to obtain the above-titledcompound [V-2].

¹ H-NMR (δ ppm; CDCl₃) 1.17 (s, 3H), 1.97 (m, 2H), 2.75 (m, 2H), 6.32(s, 1H), 7.38 (d, 2H, J=8.70 Hz), 7.48 (d, 2H, J=8.70 Hz) IR (νmax(cm⁻¹), KBr):1655, 1615, 1595

[2] Preparation of (2,2-dimethyl)-5-(4-chlorophenyl)-cyclohexane-1-one[IV-2]

10% palladium carbon was added to 5.06 g (21.6 mmole) of the compound[V-2] obtained in the Example 6[1], and ethanol was gently added theretoin an ice bath. Then, hydrogen gas was introduced at room temperature.The mixture was stirred overnight, the reaction solution was thenfiltered, and the filtrate was dried under reduced pressure. The residuewas purified using silica gel chromatography to obtain a yellow oilyproduct (4.72 g). The oily product (3.94 g) was placed in a 100 mleggplant type flask, and 17 ml of acetone was added. Then, the whole wasstirred. 3 ml of Jone's reagent was added thereto in an ice bath. After90 minutes, the reaction was ceased by adding 20 ml of ice water.Extraction was performed with diethyl ether (50 ml×2). The ether layerswere washed with a saturated saline solution, dried over anhydroussodium sulfate and dried under reduced pressure. The residue waspurified by silica gel chromatography (n-hexane:ethyl acetate=10:1) toobtain the above-titled compound [IV-2] (2.40 g).

Further, the above-titled compound [IV-2] or the compound [IV-1] wereable to be produced by the following process:

6.32 g (33 mmole) of 1-bromo-4-chlorobenzene was dissolved in 30 ml ofdried tetrahydrofuran and the solution was stirred in an argonatmosphere at -78° C. 20.6 ml (33 mmole) of an n-hexane solution ofn-butyl lithium was slowly added to the solution. After stirred for 10minutes, 1.34 mg (15 mmole) of copper cyanide was added. The whole wasstirred at -78° C. for 40 minutes, whereupon the copper cyanide wassubstantially dissolved. Further, 5 ml of tetrahydrofuran was added,whereupon the reaction solution turned transparent. 1.86 g (15 mmole) of2,2-dimethyl-5-cyclohexene-1-one was slowly added dropwise to thesolution at -78° C. in an argon atmosphere. The reaction solutionchanged in color from a light orange to a light yellow and the startingmaterial disappeared. After stirring at -78° C. for 30 minutes, asaturated aqueous solution of ammonium chloride was added. Thetemperature of the reaction solution was raised from -78° C. to 0° C.,and the crude product was extracted with diethyl ether (100 ml×1 and 50ml×1). The ether layers were washed with a saturated saline solution (20ml×1), dried over anhydrous sodium sulfate. The solvent was evaporatedunder reduced pressure to obtain the crude product (2.63 g). The crudeproduct was purified using silica gel chromatography to obtain theabove-titled compound [IV-2] (2.3 g) by n-hexane: ethyl acetate (10:1).When 1-bromo-4-fluorobenzene was used instead of1-bromo-4-chlorobenzene, the compound [IV-1] was obtained. Thephysicochemical data of the above-titled compound [IV-2] is as follows:¹ H-NMR (67ppm; CDCl₃) 1.21 (s, 3H), 1.24 (s, 3H), 1.69 (m, 1H), 1.85(m, 1H), 1.91 (m, 1H), 1.99 (m, 1H), 2.47 (m, 1H), 2.71 (dd, 1H, J=1.37,12.83 Hz), 2.95 (m, 1H), 7.15 (d, 2H, J=8.25 Hz) IR (νmax, cm⁻¹, KBr):1710, 1510, 1150

[3] Preparation of(t-(+))-(2,2-dimethyl)-5-(4-chlorophenyl)-cyclohexane-1,1'-oxolane[III(t-(+))-5] and(t-(-))-(2,2-dimethyl)-5-(4-chlorophenyl)-cyclohexane-1,1'-oxolane[III(t-(-))-6]

Dimethyl sulfoxide (2 ml) and 3 ml of dried tetrahydrofuran were addedto 166.8 mg of sodium hydride (6.95 mmole) which had been washed withn-hexane, and the mixture was stirred at room temperature. A solution oftrimethylsulfonium iodide (1.42 g, 6.95 mmole) dissolved in 4 ml ofdimethyl sulfoxide was added in an ice bath. Further, a solution of1.097 g (4.63 mmole) of the compound [IV-2] obtained in the Example 6[2]dissolved in 2 ml of dimethyl sulfoxide was added dropwise. The wholewas washed with 2 ml of dimethyl sulfoxide. The mixture was stirredovernight, and the reaction was ceased with ice water. Extraction wasperformed with diethyl ether (50 ml×3). The ether layers were washedwith a saturated saline solution, and dried over dried magnesiumsulfate, and further dried under reduced pressure. The residue waspurified by silica gel chromatography (n-hexane:ethyl acetate=20:1) toobtain the above-titled compounds [III(t-(+))-5] and [III(t-(-))- 6] asa racemic mixture.

[4] Preparation of(2,2-dimethyl)-5S-(4-chlorophenyl)-1R-(1H-2,4-triazole-1-ylmethyl)cyclohexane-1-ol[I(t-(+))-5]

A sodium salt of 1,2,4-triazole (301.1 mg, 3.3 mmole) was added to 552.9mg (2.2 mmole) of the racemic mixture obtained in the Example 6[3]. 6 mlof dimethyl sulfoxide was added thereto, and the mixture was stirred inan oil bath at 80° C. in an argon atmosphere. After 5 hours, 20 ml ofdistilled water was added to cease the reaction. The crude product wasextracted with diethyl ether (50 ml×4). The obtained ether layers werewashed with a saturated saline solution, dried over anhydrous sodiumsulfate, and further dried under reduced pressure. The residue waspurified with silica gel chromatography (ethyl acetate) to obtain aracemic mixture. The resulting racemic mixture was separated bychiracell OD (moving phase=n-hexane containing 2% isopropyl alcohol; 1ml/min) to obtain the above-titled compound [I(t-(+))-5] (25.4 mg) inthe second peak.

Melting point=173° to 175° C. Optical rotation (c=0.5; methylalcohol)=+65° C. Chemical purity: 98.0%

Example 7 [1] Preparation of(2,2-dimethyl)-5R-(4-chlorophenyl)-1S-(1H-1,2,4-triazole-1-ylmethyl)cyclohexane-1-ol[I(t-(-))-6]

The racemic mixture obtained in the Example 6 [4] was separated usingchiracell OD (moving phase=n-hexane containing 2% isopropyl alcohol) toobtain the above-titled compound [I(t-(-))-6] (31.4 mg) in the firstpeak.

Melting point=176° to 177° C. Optical rotation (c=0.5; methylalcohol)=-64° Chemical purity=99.9%

Example 8 [1] Preparation of(c-(-))-(2,2-dimethyl)-5-(4-chlorophenyl)-cyclohexane-1,1'-oxolane[III(c-(-))-7] and(c-(+))-(2,2-dimethyl)-5-(4-chlorophenyl)-cyclohexane-1,1'-oxolane[III(c-(+))-8]

3 ml of dimethyl sulfoxide was added to 72 mg (3 mmole) of sodiumhydride which had been washed with n-hexane, and the mixture was stirredat room temperature. 660.2 mg (3 mmole) of trimethylsulfoxonium iodidewas added to the mixture, and then, 5 mg (2.0 mmole) of the compound[IV-2] obtained in the Example 6[2] dissolved in 2 ml of dimethylsulfoxide was added dropwise. The mixture was washed with 1 ml ofdimethyl sulfoxide. The mixture was stirred overnight, and the reactionwas ceased with 10 ml of cold water. The crude product was extractedwith dimethyl ether (50 ml×3), and the extracts were dried under reducedpressure. The resulting residue was purified by silica gelchromatography (n-hexane:ethyl acetate=20:1) to obtain the above-titledcompounds [III(c-(+))-7] and [III(c-(+))-8] as a racemic mixture.

[2] Preparation of (2,2-dimethyl)-5S-(4-chlorophenyl)-1S-(1H-1,2,4-triazole-1-ylmethyl)cyclohexane-1-ol [I(c-(-))-7]

A sodium salt of 1,2,4-triazole (301.1 mg, 3.3 mmole) was added to 552.9mg (2.2 mmole) of the racemic mixture obtained in the Example 8[1], anddimethyl sulfoxide (6 ml) was further added. Then, the mixture wasstirred in an oil bath at 80° C. in an argon atmosphere. After 5 hours,20 ml of distilled water was added to cease the reaction. The crudeproduct was extracted with diethyl ether (50 ml×4). The obtained etherlayers were washed with a saturated saline solution, dried overanhydrous sodium sulfate, and further dried under reduced pressure. Theresidue was treated with silica gel chromatography (100% ethyl acetate)to obtain a purified racemic mixture. The resulting racemic mixture waspurified by chiracell OD (moving phase; n-hexane containing 20%isopropyl alcohol, 1.0 ml/min) to obtain the above-titled compound[I(c-(-))-7] (33.1. mg) in the second peak.

Melting point=164° to 164.5° C. Optical rotation (c=0.5; methylalcohol)=-76.2° Chemical purity=99.9%

Example 9 [1] Preparation of(2,2-dimethyl)-5R-(4-chlorophenyl)-1R-(1H-1,2,4-triazole-1-ylmethyl)cyclohexane-1-ol[I(c=(+))-8]

The racemic mixture obtained from the Example 8[2] was separated usingchiracell OD under the conditions same as those in the Example 8[2] toobtain the above-titled compound [I(c-(+))-8] (31.4 mg) in the firstpeak.

Melting point=163° to 164° C. Optical rotation (c=0.5; methylalcohol)=+73.2° Chemical purity=99.9%

The following pharmacological tests were performed using the compoundsof the present invention prepared in the Examples 1 to 9. Hereinafter,the compounds of the present invention prepared in the Examples 1 to 9will be abbreviated as shown in the following Table 1:

                  TABLE 1                                                         ______________________________________                                        Compounds of                                                                  present invention                                                                             Abbreviation                                                  ______________________________________                                        I(t-(+))-1      Compound 1                                                    I(t-(-))-2      Compound 2                                                    I(c-(-))-3      Compound 3                                                    I(c-(+))-4      Compound 4                                                    I(t-(+))-5      Compound 5                                                    I(t-(-))-6      Compound 6                                                    I(c-(-))-7      Compound 7                                                    I(c-(+))-8      Compound 8                                                    ______________________________________                                    

Example 10 Acute Toxicity

The acute toxicity by forced oral administration was investigated usingICR-JCL mice [male (M) and female (F)]. The compounds 1 to 8 accordingto the present invention were dissolved or dispersed inpolyethyleneglycol 200 or a physiological saline solution. Afteradjusted to predetermined dose, the solutions or dispersions wereadministered by a syringe or stomach sonde. After administration, thesymptoms of poisoning were continuously observed. The LD₅₀ value wasdetermined from the rate of deaths up to the seventh day. The resultsare shown in Table 2. As the comparative compound, Fluconazol (tradename; hereinafter referred to as FCZ) was used.

                  TABLE 2                                                         ______________________________________                                        Tested compounds   LD.sub.50 value (mg/kg)                                    ______________________________________                                        Compound 1         >500      (M, F)                                           Compound 2         >500      (M, F)                                           Compound 3         >500      (M, F)                                           Compound 4         >500      (M, F)                                           Compound 5         >500      (M, F)                                           Compound 6         >500      (M, F)                                           Compound 7         >500      (M, F)                                           Compound 8         >500      (M, F)                                           FCZ                1388, 1271                                                                              (M, F)                                           ______________________________________                                    

Example 11 Aromatase Activity

The aromarase activity was measured based on the method of Covey et al.[Covey, D. F. et al., BBRC, 157, (1), 81 to 86, 1988]. The aromaraseinhibitory activity was determined from the 50% inhibitory concentration(IC₅₀ value) of the enzyme activity.

That is, human placental microsome was used as the aromarase enzymesource and [19-¹⁴ C]4-androstene-3,17-dione was used as the substrate.The radioactivity of the H¹⁴ COOH released in the reaction solution as aresult of aromatization was measured to find the enzyme activityradiometrically. Then, a graph of the concentrations of the testedcompounds versus the inhibition of the enzyme activity was prepared andthe IC₅₀ values were found on the graph.

More particularly, a reaction was carried out for 30 minutes in areaction solution of 0.5 ml of 67 mM phosphate buffer (pH 7.2) whileshaking at 37° C., in a system comprised of [19-¹⁴C]4-androstene-3,17-dione (1×10⁻⁶ M, 2 kBq/ml), human placenta microsome(0.1 mg/ml protein concentration), coenzyme NADPH (2×10-³ M),glucose-6-phosphate (4×10⁻³ M), and glucose-6-phosphate dehydrogenase (4U/ml). The compounds to be tested were added as a solution in dimethylsulfoxide (final concentration of dimethyl sulfoxide=0.1 to 0.55%). TheH¹⁴ COOH released in the reaction solution was recovered in the aqueousphase by adding 5 ml of chloroform to the reaction solution at the timeof the cessation of the reaction and stirring. 0.1 ml of the aqueousphase was taken and mixed with 4 ml of liquid scintillation cocktail[Atomlight (Dupont)] to measure the radioactivity. The results are shownin Table 3. For comparison, the comparative compound (FCZ) same as inExample 10 was used.

                  TABLE 3                                                         ______________________________________                                        Tested compounds                                                                             ID.sub.50 value (μmol/l)                                    ______________________________________                                        Compound 1     >10.sup.-6                                                     Compound 2     >10.sup.-6                                                     Compound 3     >10.sup.-6                                                     Compound 4     >10.sup.-6                                                     Compound 5     >10.sup.-6                                                     Compound 6     >10.sup.-6                                                     Compound 7     >10.sup.-6                                                     Compound 8     >10.sup.-6                                                     FCZ            >10.sup.-5                                                     ______________________________________                                    

Example 12 Minimum Inhibitory Concentration

The minimum inhibitory concentrations (MIC) in vitro of the compounds 1to 8 of the present invention against microorganisms belonging to thegenera Candida, Cryptococcus, Aspergillus, and Tricophyton weredetermined. As a comparative compound, FCZ was used.

(1) The strains of the microorganisms used were as follows. Theinoculation solutions were prepared at a concentration of 1×10⁶cells/ml.

1) Candida albicans IFO 1060

2) Candida albicans IFO 1270

3) Candida albicans ATCC 762

4) Candida tropicaris IFO 1400

5) Candida krusei IFO 1395

6) Candida parapsilosis IFO 1396

7) Aspergillus fumigatus IFO 5844

8) Aspergillus fumigatus IFO 9733

9) Cryptococcus neoformans 356

10) Tricophyton mentagrophytes TIMM 1189

11) Tricophyton mentagrophytes IFO 5812

12) Tricophyton rubrum TIMM 1216

13) Tricophyton rubrum IFO 9185

(2) As the medium, a Sabouraud dextrose agar medium (Difco: dextrose 2%,agar 1.8%, not adjusted in pH) was used. The samples of the compounds tobe tested were used after dissolved in dimethyl sulfoxide. Theconcentrations of the samples were from 0.1 to 50 μ/ml in the case ofthe activity of the strains belonging to the genus Tricophyton, and werefrom 6.25 to 400/ml in the case of the activity of the strains belongingto the genera Candida, Cryptococcus, and Aspergillus.

(3) Procedure

The agar media containing the different concentrations of the compoundsto be tested were inoculated with the microorganisms, using amicroplanter (Sakuma Seisakusho). The microorganisms belonging to thegenera Candida and Cryptococcus were cultured at 27° C. for 3 days, themicroorganisms belonging to the genus Aspergillus at 27° C. for 5 days,and the microorganisms belonging to the genus Tricophyton at 27° C. for7 days. The minimum concentration on the agar medium exhibitinginhibition of growth was used as the minimum inhibitory concentration(MIC). The results are shown in Table 4.

                                      TABLE 4                                     __________________________________________________________________________    MIC of Present Compounds Against Various Fungi (μg/ml)                     Fungi                                                                         Compound                                                                            1   2   3   4    5    6    7    8    9   10  11  12  13                 __________________________________________________________________________    1     50  50  25  100  400  200  200   400<                                                                              50  50  50  50  50                 2     50  50  50  100  100     6.25                                                                            100   400<                                                                              25  25  25  25  25                 Racemic                                                                             50  25  50  100  200  25   100   400<                                                                              50  50  50  50  50                 mixture of                                                                    1 and 2                                                                       3     50  100 50   200<                                                                               200<                                                                               200<                                                                               200<                                                                               400<                                                                              50   50<                                                                               50<                                                                              50  50                 4     50  100 50   200<                                                                               200<                                                                               200<                                                                              200  400  50   50<                                                                               50<                                                                              50  50                 Racemic                                                                             50  100 50   200<                                                                               200<                                                                               200<                                                                               200<                                                                               400<                                                                              100  50<                                                                               50<                                                                              50  50                 mixture of                                                                    3 and 4                                                                       5     25  25  25  100  200  25     12.5                                                                               12.5                                                                             25  50  50  50  50                 6     25  25    12.5                                                                            100  100    12.5                                                                                6.25                                                                               6.25                                                                              12.5                                                                            25  50  25  25                 Racemic                                                                             25  25  25  100  200  25     12.5                                                                               12.5                                                                               12.5                                                                            50  50  50  50                 mixture of                                                                    5 and 6                                                                       7     25    12.5                                                                            25    50<                                                                                50<                                                                              25    50  50   25  50  25  50  50                 8     25    12.5                                                                              12.5                                                                              50<                                                                                50<                                                                              50    50  50   25   50<                                                                               50<                                                                               50<                                                                               50<               Racemic                                                                             25    12.5                                                                            25    50<                                                                                50<                                                                              50    50  50   25   50<                                                                               50<                                                                               50<                                                                               50<               mixture of                                                                    7 and 8                                                                       FCZ    400<                                                                              400<                                                                              400<                                                                              400<                                                                              200    12.5                                                                              400<                                                                               400<                                                                              25   400<                                                                              400<                                                                              400<                                                                              400<              __________________________________________________________________________

Example 13 Preparation of Capsules

The compound 1 (100 mg) of the present invention, 50 mg ofpolyoxyethylene sorbitamine monooleate, and 250 mg of starch werethoroughly mixed and filled in capsules to prepare capsules.

INDUSTRIAL APPLICABILITY

The novel azole derivative according to the present invention is low intoxicity, has an antifungal activity, and has an aromarase inhibitoryactivity (therefore, an antitumor activity). Further, such activitiesare remarkable when continuously administered.

We claim:
 1. A method of inhibiting aromatase, comprising administeringto a mammal in need thereof a pharmaceutical composition comprising aneffective aromatase inhibiting amount of a compound of formula (I):##STR10## wherein X₁ is a chlorine or fluorine atom, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 2. A method according to claim 1, wherein a racemicmixture of compounds of the formulas [I(t-(+))] and [I(t-(-))] areadministered: ##STR11## wherein X₁ is a chlorine or fluorine atom, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 3. A method according to claim 1, wherein a compoundof the formula [I(t-(+))] is administered: ##STR12## wherein X₁ is achlorine or fluorine atom, or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier.
 4. A methodaccording to claim 1, wherein a compound of the formula [I(t-(-))] isadministered: ##STR13## wherein X₁ is a chlorine or fluorine atom, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 5. A method according to claim 1, wherein a racemicmixture of compounds of the formulas [I(c-(+))] and [I(c-(-))] areadministered: ##STR14## wherein X₁ is a chlorine or fluorine atom, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 6. A method according to claim 1, wherein a compoundof the formula [I(c-(+))] is administered: ##STR15## wherein X₁ is achlorine or fluorine atom, or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier.
 7. A methodaccording to claim 1, wherein a compound of the formula [I(c-(-))] isadministered: ##STR16## wherein X₁ is a chlorine or fluorine atom, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 8. A method according to claim 1, wherein a tumor inthe mammal is treated.
 9. A method according to claim 2, wherein a tumorin the mammal is treated.
 10. A method according to claim 3, wherein atumor in the mammal is treated.
 11. A method according to claim 4,wherein a tumor in the mammal is treated.
 12. A method according toclaim 5, wherein a tumor in the mammal is treated.
 13. A methodaccording to claim 6, wherein a tumor in the mammal is treated.
 14. Amethod according to claim 7, wherein a tumor in the mammal is treated.